Borrelia burgdorferi, the infectious agent of Lyme disease, is transmitted to mammals through the bite of infected Ixodes ticks. Our broad objective is to use a genetic approach to elucidate the molecular mechanisms of adaptation and variation in B. burgdorferi and their roles in the infectious cycle. Most genetic methods that have been developed for other bacteria can not be directly applied to B. burgdorferi. We previously developed a method of gene inactivation by allelic exchange. We recently developed additional genetic tools that have greatly facilitated our efforts to inactivate genes, introduce foreign DNA and improve methods of transformation in B. burgdorferi. These include two antibiotic resistance genes, whose properties provide much greater utility than the original selectable marker. The availability of additional genetic markers has also permitted more detailed studies, such as complementation analyses and the generation of strains with double mutations. These improved markers have facilitated ongoing studies to address the feasibility of conjugation with Escherichia coli as a method to more efficiently introduce DNA into B. burgdorferi. They have also allowed us to demonstrate exchange of genetic material between cultured spirochetes that are distinguishable by their resistance phenotypes, and to investigate a recently described method to create random, tagged mutations. Perhaps most significantly, the improved selectable markers have directly contributed to the development of a shuttle vector for stable introduction of autonomously replicating DNA in both E. coli and B. burgdorferi. This is a standard and important genetic tool that was previously lacking for studies with B. burgdorferi. In addition to its utility as a genetic tool, the shuttle vector is currently being used to investigate plasmid structure and function in B burgdorferi. These studies have resulted in the definition of a B. burgdorferi plasmid region conferring autonomous replication and maintenance functions. This represents the first identification of a plasmid origin of replication in borrelia. The shuttle vector is also being used to explore structural relationships between linear and circular plasmids in B. burgdorferi An equally significant recent advance was the demonstration of the first gene inactivation by allelic exchange in infectious spirochetes. Previously, all successful gene inactivations were made in an attenuated, non-infectious variant of the type strain B31, which is more readily transformed by electroporation., We have been able to overcome this impediment by careful optimization of electroporation conditions with the recently developed selectable markers. Gene inactivation, targeted insertion and transformation with the shuttle vector have now all been performed in infectious isolates. However, while possible, genetic manipulation of infectious B. burgdorferi is still inefficient and not routine. Ongoing efforts are directed towards improving existing techniques and investigating additional methods with which to transform pathogenic B. burgdorferi. All of these recent advances have brought us closer to our goal of analyzing the contributions of specific spirochetal genes to transmission, infection and disease.